Abstract Breast cancer remains the leading cause of cancer death among women worldwide, rendering conventional therapies insufficient despite decades of research. Despite originally considered an immunologically silent malignancy, recent studies have shown encouraging results, from treatment of breast cancer with checkpoint inhibition to enhance the immunogenicity and tumor cell killing mediated by immune cells. The epitope landscape in breast cancer is minimally described; thus it is necessary to identify T-cell targets to develop cancer-specific immune-mediated therapies. This project investigates four proteins commonly upregulated in breast cancer and thus probable tumor-associated antigens (TAAs), namely, aromatase, prolactin, never in mitosis a related kinase 3 (NEK3), and protein inhibitor of activated STAT3 (PIAS3). Each contributes to increase growth, survival, and motility of malignant cells. To uncover novel epitopes for cytotoxic T-cells, a reverse immunology approach is applied. In silico screening via NetMHC was used to predict peptides within the full length of each of the four proteins that bind to HLA-A*0201 and HLA-B*0702. Via in silico screening 415 HLA-A*0201 or HLA-B*0702 binding peptides were predicted An MHC ELISA was applied to experimentally confirm which of the peptides are true HLA-A*0201 and HLA-B*0702 binders, reducing the library from 415 to 147. The 147 peptides were evaluated for T-cell recognition utilizing DNA barcode labeled MHC multimers to screen peripheral blood lymphocytes from 24 breast cancer patients and 18 healthy donor samples. Significantly more TAA specific T-cell responses were detected in breast cancer patients than healthy donors for both HLA-A*0201 (p=0.0039) and HLA-B*0702 (p<0.001) restricted peptides. Thus, the inspected proteins aromatase, prolactin, NEK3 and PIAS3 indeed contain targets for T-cell reactivity. Importantly, several of the responses were identified towards peptides with a predicted intermediate HLA-binding affinity. This emphasizes the importance of including such weaker affinity ligands in the search for epitopes within shared TAAs. Citation Format: Nadia Viborg Petersen, Sofie Ramskov, Rikke Sick Andersen, Özcan Met, Per thor Straten, Amalie Kai Kai Bentzen, Sine Reker Hadrup. T-cell recognition of breast cancer antigens [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B036.